US2019233844A1PendingUtilityA1

Spatiotemporal regulators

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Assignee: SENTI BIOSCIENCES INCPriority: Jul 26, 2016Filed: Jul 26, 2017Published: Aug 1, 2019
Est. expiryJul 26, 2036(~10 yrs left)· nominal 20-yr term from priority
Inventors:Timothy Kuan-Ta LuRemus Wong
C12N 15/63C12N 15/62C12N 2830/008C12N 2840/105C12N 2310/141C12N 9/22C12N 2830/30C12N 5/0607C12N 15/85C12N 15/67C12N 2310/3513C12N 2310/122C12N 2310/14C12N 2800/30C12N 5/0606C12N 2310/20C12N 15/65C12N 15/113C12N 15/111C12N 15/86C12Y 301/21C12N 2800/24C12N 5/0637
53
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Claims

Abstract

Provided herein, in some embodiments, are methods, compositions, systems and kits that enable spatiotemporal regulation of nucleic acid expression in engineered cells.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . An engineered genetic construct comprising at least one synthetic promoter that has higher activity in target cells relative to non-target cells and is operably linked to (a) a nucleotide sequence encoding a product of interest and (b) a 3′ untranslated region (UTR) comprising at least one microRNA (miRNA) sensor that includes at least one miRNA binding site to which at least one miRNA binds, wherein the at least one miRNA is inactive or active at a low level in the target cells, and wherein the at least one miRNA is active in non-target cells at a level detectable by the miRNA sensor. 
     
     
         2 . The engineered genetic construct of  claim 1 , wherein the microRNA sensor includes at least two miRNA binding sites. 
     
     
         3 . The engineered genetic construct of  claim 2 , wherein the microRNA sensor includes 2-10 miRNA binding sites. 
     
     
         4 . The engineered genetic construct of  claim 3 , wherein the microRNA sensor includes 5-10 miRNA binding sites. 
     
     
         5 . The engineered genetic construct of  claim 2 , wherein the microRNA sensor includes at least five miRNA binding sites. 
     
     
         6 . The engineered genetic construct of  claim 5 , wherein the microRNA sensor includes 5-10 miRNA binding sites. 
     
     
         7 . The engineered genetic construct of any one of  claims 2 - 6 , wherein the miRNA binding sites are located in tandem. 
     
     
         8 . The engineered genetic construct of any one of  claims 2 - 7 , wherein the miRNA binding sites are identical to each other. 
     
     
         9 . The engineered genetic construct of any one of  claims 1 - 8 , wherein the 3′ UTR comprising at least two miRNA sensors, each specific to a different miRNA. 
     
     
         10 . The engineered genetic construct of any one of  claims 1 - 9 , wherein the at least one miRNA (miR) is selected from miR-154, miR-497, miR-29A, miR-720, miR-205, miR-494, miR-224, miR-191, miR-21, miR-96, miR-449A and miR-183. 
     
     
         11 . The engineered genetic construct of any one of  claims 1 - 10 , wherein the target cells are cancerous cells, immune cells, or neurons. 
     
     
         12 . The engineered genetic construct of any one of  claims 1 - 11 , wherein the synthetic promoter has a length of 100-500 nucleotides. 
     
     
         13 . The engineered genetic construct of  claim 12 , wherein the synthetic promoter has a length of 100-125 nucleotides. 
     
     
         14 . The engineered genetic construct of any one of  claims 1 - 13 , wherein the synthetic promoter includes tandem repeat nucleotide sequences. 
     
     
         15 . The engineered genetic construct of  claim 14 , wherein the length of each of the nucleotide sequences is less than 12 nucleotides. 
     
     
         16 . The engineered genetic construct of  claim 14  or  15 , wherein the synthetic promoter includes 2-20 tandem repeat nucleotide sequences. 
     
     
         17 . The engineered genetic construct of any one of  claims 1 - 16 , wherein a nucleotide spacer is positioned between each of the repeat nucleotides sequences. 
     
     
         18 . The engineered genetic construct of any one of  claims 1 - 17 , wherein the activity of the synthetic promoter is at least 10% higher in the target cells relative to non-target cells. 
     
     
         19 . The engineered genetic construct of  claim 18 , wherein the activity of the synthetic promoter is at least 50% higher in the target cells relative to non-target cells. 
     
     
         20 . The engineered genetic construct of  claim 19 , wherein the activity of the synthetic promoter is at least 100% higher in the target cells relative to non-target cells. 
     
     
         21 . The engineered genetic construct of any one of  claims 1 - 19 , wherein the product of interest is a therapeutic molecule, a prophylactic molecule and/or a diagnostic molecule. 
     
     
         22 . The engineered genetic construct of any one of  claims 1 - 21 , wherein the product of interest is a protein, peptide or nucleic acid. 
     
     
         23 . The engineered genetic construct of  claim 22 , wherein the product of interest is a nucleic acid selected from RNA, DNA or a combination of RNA and DNA. 
     
     
         24 . The engineered genetic construct of  claim 23 , wherein product of interest is a RNA selected from short-hairpin RNAs, short interfering RNAs and micro RNAs. 
     
     
         25 . The engineered genetic construct of  claim 21 , wherein the product of interest is a therapeutic and/or prophylactic molecule selected from antibodies, enzymes, hormones, inflammatory molecules, anti-inflammatory molecules, immunomodulatory molecules, and anti-cancer molecules. 
     
     
         26 . The engineered genetic construct of  claim 21 , wherein the product of interest is a diagnostic molecule selected from fluorescent molecules and luminescent molecules. 
     
     
         27 . A vector comprising the engineered genetic construct of any one of  claims 1 - 26 . 
     
     
         28 . A cell comprising the engineered genetic construct of any one of  claims 1 - 26  or the vector of  claim 27 . 
     
     
         29 . A composition comprising the engineered genetic construct of any one of  claims 1 - 26 , the vector of  claim 27 , or the cell of  claim 28 . 
     
     
         30 . A kit comprising an engineered genetic construct comprising at least one synthetic promoter that has higher activity in target cells relative to non-target cells and is operably linked to a 3′ untranslated region (UTR) comprising at least one microRNA (miRNA) sensor that includes at least one miRNA binding site to which at least one miRNA binds, wherein the at least one miRNA is inactive or active at a low level in the target cells, and wherein the at least one miRNA is active in non-target cells at a level detectable by the miRNA sensor, wherein the construct further comprises restriction sites located between the promoter and the 3′ UTR. 
     
     
         31 . A method comprising delivery to a cell the engineered genetic construct of any one of  claims 1 - 26  or the vector of  claim 27 . 
     
     
         32 . A method comprising delivery to a subject the engineered nucleic acid of any one of  claims 1 - 26  or the vector of  claim 26 . 
     
     
         33 . The method of  claim 31 , wherein the subject is a human subject.

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